Longitudinal monitoring of cytoplasmic RBP-RNA interactions and transcriptome in living cells by engineered protein nanocages

Most existing RNA sequencing methods rely on cell lysis or fixation, limiting their use in longitudinal studies of the same cell population. Here, we introduce POND-seq (protein nanocage-empowered non-destructive sequencing), a strategy that employs secretory protein nanocages fused to RNA-binding proteins (RBPs) to recover RBP-associated RNAs from living cells. POND-seq robustly identifies RNA targets of cytoplasmic RBPs across multiple cell types and enables longitudinal tracking of dynamic changes in RBP-associated RNA profiles under stress conditions. Fusion to poly(A)-binding protein C (PABPC1) further allows monitoring of transcriptomic responses and selectively profiles cell-type-specific transcriptomes from mixed populations without cell dissociation and sorting. Additionally, POND-seq supports functional interrogation of RBP domains and residues involved in RNA association and enables scalable analysis of RBP variants, as demonstrated by a systematic assessment of disease-associated fragile X messenger ribonucleoprotein 1 (FMR1) mutations. Together, POND-seq provides a versatile and scalable platform for non-destructive and longitudinal analysis of cytoplasmic transcriptomes and RBP-associated RNAs.